Evaluation of binding capacity of circulating insulin-like growth factor binding protein-1b in salmonids using a ligand immunofunctional assay.

Insulin-like growth factor-binding proteins (IGFBPs) regulate the activity of insulin-like growth factor (IGF)-1. Among the three major circulating IGFBPs in salmonids, IGFBP-1b is an inhibitor of IGF activity induced under catabolic conditions. IGFBP-1b is considered to quickly sequester IGF-1 from the circulation. However, the level of circulating IGFBP-1b present in its unoccupied free form is unknown. Here, we aimed to develop a non-equilibrium ligand immunofunctional assay (LIFA) to evaluate IGF-binding capacity of circulating intact IGFBP-1b. Purified Chinook salmon IGFBP-1b, its antiserum, and europium-labeled salmon IGF-1 were used as the assay components. In the LIFA, IGFBP-1b was first captured by the antiserum, allowed to bind to the labeled IGF-1 for 22 h at 4 °C, and quantified its IGF-binding capacity. Serial dilutions of the standard and serum were prepared simultaneously within a certain concentration range (1.1-12.5 ng/ml). In underyearling masu salmon, IGF-binding capacity of intact IGFBP-1b was higher in fasted fish than in fed fish. Transferring Chinook salmon parr to seawater also increased IGF-binding capacity of IGFBP-1b, most likely due to osmotic stress. In addition, there was a strong relationship between total IGFBP-1b levels and its IGF-binding capacity. These results suggest that IGFBP-1b expressed under stress is mostly present in the free form. On the contrary, during smoltification of masu salmon, IGF-binding capacity of IGFBP-1b in the serum was relatively low and less related to the total IGFBP-1b level, suggesting its functional difference under certain physiological conditions. These results indicate that estimating both total IGFBP-1b level and its IGF-binding capacity is useful for evaluating the catabolic status and unraveling the regulation of IGF-1 activity by IGFBP-1b.